8-Nitro-cGMP Attenuates the Interaction between SNARE Complex

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Letter

8-Nitro-cGMP attenuates the interaction between SNARE complex and complexin through S-guanylation of SNAP-25 Yusuke Kishimoto, Kohei Kunieda, Atsushi Kitamura, Yuki Kakihana, Takaaki Akaike, and Hideshi Ihara ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00363 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 8, 2017

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ACS Chemical Neuroscience

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Title: 8-Nitro-cGMP attenuates the interaction between SNARE complex and 6 7

complexin through S-guanylation of SNAP-25 8 9 10 12

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Author List: 14

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Yusuke Kishimoto1, Kohei Kunieda1, 2, Atsushi Kitamura1, Yuuki Kakihana 1, Takaaki 16

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Akaike3, Hideshi Ihara1* 17 18 20

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Department of Biological Science, Graduate School of Science, Osaka Prefecture

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University, Sakai, Osaka, Japan; 2Department of Protein Factory, Translational Research 25

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Center, Fukushima Medical University, Fukushima, Fukushima, Japan; 3Department of 27

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Environmental Health Science and Molecular Toxicology, Tohoku University Graduate 29

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School of Medicine, Sendai, Miyagi, Japan 30 31 32 3

*Corresponding author: Hideshi Ihara, Department of Biological Science, Graduate 34 36

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School of Science, Osaka Prefecture University, Address: #608 Bldg.C10, 1-1 38

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Gakuen-cho, Nakaku, Sakai, Osaka, 599-8531, Japan, Tel: +81-72-254-9753, E-mail: 40

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[email protected], 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 59

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60 ACS Paragon Plus Environment


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Abstract: 6 7

8-Nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) is the second 8 10

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messenger in nitric oxide/reactive oxygen species redox signaling. This molecule 12

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covalently binds to protein thiol groups, called S-guanylation, and exerts various 14

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biological functions. Recently, we have identified synaptosomal-associated protein 25 16

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(SNAP-25) as a target of S-guanylation, and demonstrated that S-guanylation of 18

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SNAP25 enhanced SNARE complex formation. In this study, we have examined the 19 20

effects of S-guanylation of SNAP-25 on the interaction between the soluble SNARE 21 23

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complex and complexin (cplx), which binds to the SNARE complex with a high affinity. 25

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Pull-down assays and co-immunoprecipitation experiments have revealed that 27

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S-guanylation of Cys90 in SNAP-25 attenuates the interaction between the SNARE 29

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complex and cplx. In addition, blue native-PAGE followed by western blot analysis 30 31

revealed that the amount of cplx detected at a high molecular weight decreased upon 32 3

8-nitro-cGMP treatment in SH-SY5Y cells. These results demonstrated for the first time 34 36

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that S-guanylation of SNAP-25 attenuates the interaction between the SNARE complex 38

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and cplx. 39 40 42

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Key words: 43 4

8-nitro-cGMP, S-guanylation, SNAP-25, SNARE complex, complexin, exocytosis 45 46 47 48 49 50 51 52 53 54 5 56 57 59

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Introduction 6 7

Nitric oxide (NO) is a gasotransmitter that is involved in both physiological 8 10

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and pathological functions, including synaptic plasticity and neurodegenerative 12

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disorders in the nervous system 1. Typically, NO exerts its multiple functions through 14

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two mechanisms 2. One is the NO/guanosine 3′,5′-cyclic monophosphate (cGMP) 16

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pathway; NO, generated by NO synthase (NOS), activates soluble guanylate cyclase to 18

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generates cGMP, which activates cGMP dependent protein kinase 2. The other is the 19 20

pathway involving chemical modification of biomolecules by NO or peroxynitrite 21 2

which is generated by the interaction of NO and reactive oxygen species (ROS) 2. 25

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Although NOSs are considered to be NO-producing enzymes, they produce not 27

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only NO but also superoxide by the uncoupling reaction 3-5, the levels of which are 29

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regulated by several mechanisms, such as phosphorylation 4 and splicing 3. ROS are 31

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well-known toxicants that damage DNA, protein, and lipids 6. However, recent studies 3

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demonstrated that ROS also work as redox signaling molecules in certain conditions 6. 34 35

NO and ROS from NOSs were both involved in the modulation of redox signaling 3, 4. 38

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Moreover, ROS play an important role in the synaptic plasticity formations 7. 40

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Superoxide produced via calcium influx through the N-methyl-D-aspartate (NMDA) 42

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receptor is necessary for long-term potentiation (LTP) in the hippocampus 8. NADPH 43 4

oxidase (Nox) subunits deficit mice show impaired LTP and hippocampus-dependent 45 46

memory 9. Furthermore, superoxide generated by Nox in activated microglia induces 47 48

long-term depression (LTD) in the hippocampus 10. 51

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These studies demonstrated the

physiological relevance of ROS in brain function. 53

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Previously, we discovered 8-nitroguanosine 3′,5′-cyclic monophosphate 5

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(8-nitro-cGMP) in the process of NO/ROS redox signaling 6, 11, 12. This molecule 56 57 59

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covalently binds to protein thiol groups (S-guanylation) and exerts various biological 7

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functions, such as cytoprotection4, 13, cell senescence 14, 15, and autophagy 16. Some 8 10

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target proteins of this post-translational modification (PTM) have been reported 12

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previously (e.g., H-Ras 14, 15, mitochondrial heat shock protein 60 17, cGMP-dependent 14

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protein kinase 18, and Kelch-like ECH-associated protein 1 (Keap1) 12, 13). We have 16

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found that synaptosomal-associated protein 25 (SNAP-25), a member of the soluble 18

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N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins, was 19 20

S-guanylated by 8-nitro-cGMP, and S-guanylation of Cys90 in SNAP-25 enhances the 21 2

formation of the SNARE complex 19. Thus, 8-nitro-cGMP is expected to function as an 25

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important mediator of NO/ROS signaling in nervous system. However, the molecular 27

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mechanism for regulation of SNAP-25 by 8-nitro-cGMP is not yet well understood. 29

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In the process of exocytosis of neurotransmitters, the SNARE complex is the 31

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key component to catalyze membrane fusion 20. The SNARE complex is composed of 32 3

three SNARE proteins (SNAP-25, syntaxin, and vesicle-associated membrane protein 2 34 35

(VAMP2)) 20. Exocytosis of neurotransmitters requires multistep reactions including 38

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“docking” and “priming,” and then, Ca2＋ influx triggers membrane fusion 21. PTM of 40

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SNAP-25 has some neuropathophysiological significance. The residue substitutions of 42

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phosphorylation sites resulted in an increase in anxiety-related behavior in mice 22 and 4

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physical stress for mice enhances SNAP-25 phosphorylation 23. 45 47

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Exocytosis of neurotransmitters is modulated not only by SNARE proteins but 49

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also by exocytosis-modulatory proteins, including synaptotagmins, mammalian 51

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uncoordinated-18 (Munc18), and complexin (cplx) 20, 21. Cplx is an 53

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exocytosis-regulatory protein that binds to the ternary SNARE complex with a high 5

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affinity but not to monomeric SNAP-25, syntaxin, and VAMP2 24. Cplx binding to the 56 57 59

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SNARE complex is an important process of priming and exocytosis 24. In addition, Cplx 7

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enhances SNARE complex oligomerization, which is required for exocytosis 25. Besides, 8 9

cplx plays important roles in brain functions 26, 27. For instance, cplx I-knockout mice 10 12

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showed motor incoordination; a reduction in grooming, rearing, and exploratory 14

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behavior; and social behavior deficit 26, 27. However, molecular mechanism regulating 16

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the cplx function has not been fully understood. 18

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To date, the effect of 8-nitro-cGMP on the interaction between the SNARE 19 20

complex and exocytosis regulatory proteins has not been clarified. In this study, we 21 23

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have investigated the effect of 8-nitro-cGMP on the interaction between the SNARE 25

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complex and cplx to clarify the roles of 8-nitro-cGMP in exocytosis in neurons. We 27

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revealed that S-guanylation of SNAP-25 by 8-nitro-cGMP attenuates the interaction 29

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between the SNARE complex and cplx. 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 59

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Results and discussion 6 7

Cell permeability of 8-nitro-cGMP in vascular smooth muscle cells has been 8 9

reported 12. Here, we confirmed whether 8-nitro-cGMP penetrated the membrane of 10 12

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SH-SY5Y neuroblastoma cells by western blotting, immunohistochemical analysis 14

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(IHC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis 16

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(Fig. 1). Western blot analysis revealed that 8-nitro-cGMP treatment of SH-SY5Y cells 18

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increased the amount of S-guanylated proteins in the cell lysates that contains both 19 20

cytosolic and membrane proteins (Fig. 1A). Next, we performed IHC to detect the 21 23

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levels of 8-nitro-cGMP and S-guanylated proteins in SH-SY5Y cells treated with 25

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8-nitro-cGMP. As shown in Fig. 1B, we observed 8-nitro-cGMP-dependent elevation of 27

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intracellular 8-nitro-cGMP and S-guanylated protein levels in the SH-SY5Y cells. 29

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Furthermore, we analyzed the concentration of intracellular 8-nitro-cGMP in SH-SY5Y 30 31

cells by LC-MS/MS using a stable-isotope dilution method. As shown in Fig. 1C, 3

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8-nitro-cGMP was detected at the same retention time as was 8- nitro-c[13C10]GMP in 34 36

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the lysate from 8-nitro-cGMP-treated cells. The concentration of 8-nitro-cGMP was 38

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determined as 6.74 ± 0.10 pmol/mg protein estimated from 8- nitro -c[13C10]GMP. The 40

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concentration of 8-nitro-cGMP in the brains of mice has been reported to be 42

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approximately 3 pmol/mg protein 19; thus, the intracellular 8-nitro-cGMP concentration 43 4

in SH-SY5Y cells treated with 100 µM 8-nitro-cGMP is not very different from that 45 47

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under physiological conditions. Taken together, our results indicate that exogenously 49

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added 8-nitro-cGMP penetrates the cell membrane of SH-SY5Y cells and reacts with 51

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the cytoplasmic proteins. 53

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We have previously reported that the S-guanylation of SNAP-25 enhances the 5

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formation of the SNARE complex 19, but its effect on the interaction between the 56 57 59

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SNARE complex and cplx has not been clarified. Here, we investigated the effect of 6 7

8-nitro-cGMP on the interaction between the SNARE complex and cplx. First, to 8 10

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examine the interaction of cplx with the SNARE complex, we performed a pull-down 12

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assay. SNARE complexes in the solubilized synaptosome (Fig. 2A and B) and cells (Fig. 14

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2C and D) were bound to exogenously added cplx conjugated with glutathione 16

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S-transferase (GST)-beads. The amount of pull-downed SNAP-25 was analyzed by 18

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western blotting (Fig. 2). Initially, we predicted that the amount of SNAP-25 harvested 19 20

by cplx in the pull-down assay would be increased by 8-nitro-cGMP treatment, because 21 2

our previous study showed that 8-nitro-cGMP enhances SNARE complex formation 19. 25

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Unexpectedly, the amount of SNAP-25 in the rat synaptosome bound to exogenously 27

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added GST-cplx was significantly decreased by 8-nitro-cGMP treatment (Fig. 2). We 29

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previously reported that the main target of S-guanylation of SNAP-25 is the cysteine 90 31

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in this protein 19. Therefore, to examine whether the effect of 8-nitro-cGMP depends on 32 3

the cysteine 90 S-guanylation of SNAP-25, we performed the pull-down assay using 34 36

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SH-SY5Y cells-transfected with FLAG-tagged SNAP-25 (wild-type or cysteine 90 38

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alanine point mutant (C90A)) (Fig. 2C, D). The substitution of cysteine 90 to alanine 40

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in SNAP-25 did not affect the binding of GST-cplx to SNARE complex. The amount of 42

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FLAG-tagged SNAP-25 bound to GST-cplx decreased upon 8-nitro-cGMP treatment in 43 4

the SNAP-25 wild-type transfected cells; on the other hand, the decrease was 45 47

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suppressed in SNAP-25 C90A mutant transfected cells (Fig. 2C, D). It has reported that 49

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cplx binds to the SNARE complex with a high affinity but does not bind to the 51

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SNAP-25 monomer. Hence, the SNAP-25 detected here is a portion of the SNARE 53

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complex. These results suggest that the S-guanylation of SNAP-25 at C90 by 5

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8-nitro-cGMP critically attenuates the interaction between the SNARE complex and 56 57 59

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exogenously added cplx. 6 7

In the pull-down assay, there is the possibility that the endogenously expressed 8 10

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cplx that has already bound to the SNARE complex inhibits the interaction between the 12

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SNARE complex and exogenously added cplx. In the next study, to examine whether 14

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8-nitro-cGMP attenuates the interaction between the SNARE complex and 16

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endogenously expressing cplx in living cells, we performed co-immunoprecipitation 18

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(co-IP) of SNAP-25 and cplx followed by western blot analysis using SH-SY5Y cells 19 20

co-transfected with FLAG-tagged SNAP-25 (wild-type or C90A mutant) and V5-tagged 21 23

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cplx (Fig. 3). 8-Nitro-cGMP treatment significantly decreased the amount of V5-tagged 25

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cplx co-immunoprecipitated by the anti-FLAG-tag antibody in SH-SY5Y cells 27

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co-transfected with FLAG-tagged SNAP-25 wild-type and V5-tagged cplx (Fig. 3). On 29

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the other hand, the decrease was suppressed in SNAP-25 C90A mutant-transfected cells 30 31

(Fig. 3). These results indicate that 8-nitro-cGMP attenuates the interaction between the 32 3

SNARE complex and not only exogenously added cplx but also endogenously 34 36

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expressing cplx in the living cells through S-guanylation of SNAP-25 at C90. 38

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In the next study, to confirm the affinity attenuation between the SNARE 40

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complex and cplx by another way, we performed blue native (BN)-polyacrylamide gel 42

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electrophoresis (PAGE) followed by western blotting to analyze the size and amount of 43 4

cplx and SNAP-25-containing protein complexes in SH-SY5Y cells (Fig. 4). BN-PAGE 45 47

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is the method to analyze protein complexes and allows isolation of protein complexes 48

while preserving their native conformation 28. Recently, this method was utilized for 51

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analyzing the SNARE complex with cplx and other SNARE modulatory proteins in the 53

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human brain of individuals with schizophrenia 29. In this study, we applied the 5

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BN-PAGE to analyze the SNARE complex, including modulatory proteins such as cplx 56 57 59

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in the SH-SY5Y cells transfected with FLAG-tagged SNAP-25 and V5-tagged cplx. To 6 7

our knowledge, this is the first report to analyze the SNARE complex formation and 8 10

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interaction of cplx in the cultured cells by BN-PAGE. As shown in Fig. 4A, SNAP-25 12

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immunoreactive bands of various sizes were observed and 8-nitro-cGMP increased the 14

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amount of high-molecular-mass complexes containing SNAP-25 (Fig. 4B), consistent 16

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with our previous finding, indicating that 8-nitro-cGMP enhances SNARE complex 18

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formation and the amount of SDS-resistant SNARE complexes 19. In addition, 19 20

8-nitro-cGMP decreased the amount of V5-tagged cplx detected at a molecular mass of 21 23

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approximately 1,000 kDa, wherein one SNAP-25 immunoreactive band was detected 25

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(Fig. 4C, D). Because our co-IP analysis in this study revealed that 8-nitro-cGMP 27

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reduced the interaction between SNAP-25 and cplx in SH-SY5Y cells (Fig. 3), 29

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V5-tagged cplx detected at a high molecular mass was presumed to be associated with 30 31

the SNARE complex. Taking the facts mentioned above into considerations, our results 32 3

strongly suggest that 8-nitro-cGMP increases the amount of SNAP-25 containing large 34 36

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complex, on the other hand, it decreases the affinity of cplx with the SNARE complex 38

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at high molecular mass. 40

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Cplx has been well-known to regulate neurotransmitter release at the synapse 42

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by binding to the SNARE complex with a high affinity 24. However, the molecular 43 4

mechanism that regulates the affinity between cplx and the SNARE complex has not 45 47

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been fully understood. PTMs of cplx and SNARE proteins are candidate mechanism for 49

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regulating the affinity between cplx and the SNARE complex. Indeed, it has previously 51

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been reported that cplx is phosphorylated at ser115 in the rat brain and that 53

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phosphorylated cplx exhibits enhanced SNARE complex binding 30. The authors 5

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speculated that the phosphorylation may provide a new route for modulating fast 56 57 59

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neurotransmitter release 30. Here, we demonstrated for the first time that the cplx 6 7

binding to the SNARE complex was modulated by PTM (S-guanylation) of SNAP-25. 8 10

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The S-guanylation of SNAP-25 attenuates the interaction between cplx and the SNARE 12

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complex. The S-guanylation of SNAP-25 may be involved in neurotransmitter release. 14

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Our present studies unclarified the neurophysiological meaning of the regulation of 16

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affinity changing between the SNARE complex and cplx by 8-nitro-cGMP. Previously, 18

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it was reported that attenuation of the interaction of the SNARE complex with cplx in 19 20

squid giant presynaptic terminals inhibited neurotransmitter release at a late prefusion 21 2

step of synaptic vesicle exocytosis 25. Thus, 8-nitro-cGMP might regulate 25

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neurophysiological function by regulating SNARE complex formation and the affinity 27

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between the SNARE complex and cplx. Further work is in progress to clarify the 29

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neurophysiological role of S-guanylation of SNAP25 by 8-nitro-cGMP. 30 31

In conclusion, 8-nitro-cGMP treatment attenuates the interaction between the 32 3

SNARE complex and both exogenously added and endogenously expressing cplx. 34 36

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Moreover, cplx was detected at a high molecular mass in SH-SY5Y cells, and it was 38

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released upon 8-nitro-cGMP treatment. Taken together, our results from a pull-down 40

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assay, co-IP, and BN-PAGE strongly suggest that 8-nitro-cGMP attenuates the 42

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interaction between the SNARE complex and cplx through S-guanylation at C90 in 43 4

SNAP-25. Our findings for 8-nitro-cGMP, which is generated through NO/ROS 45 47

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signaling, described above, provide new insights into NO/ROS redox signaling in 49

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neurotransmission. Because the binding of cplx to the SNARE complex is an important 51

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process in exocytosis, there is a possibility that 8-nitro-cGMP regulates exocytosis, 53

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neurotransmission, and brain functions. 54 5 56 57 59

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Methods: 6 7

Materials 8 10

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SH-SY5Y cells were kindly supplied by Dr. Hidemitsu Nakajima (Osaka 12

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Prefecture University). The anti-8-nitro-cGMP antibody (clone 1G6) 12, 14

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anti-S-guanylated protein antibody 12, and anti-SNAP-25 antibody (clone BR05) 19 were 16

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prepared as previously described. pcDNA3.1/nV5-DEST, LR reaction kit, 18

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NativePAGETM Kit were from Thermo Fisher Scientific (Waltham, MA). Glutathione 19 20

(GSH) sepharose, polyvinylidene difluoride (PVDF) membrane, peroxidase 21 23

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(POD)-conjugated secondary antibodies were from GE Healthcare (Buckinghamshire, 25

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England, UK). Dulbecco's Modified Eagle's Medium (D-MEM) and Hoechst33258 27

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were purchased from Wako Pure Chemical (Osaka, Japan). Penicillin-Streptomycin (PS) 29

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solution, protease inhibitor cocktail, Blocking One and anti-V5-tag antibody were from 30 31

Nacalai Tesque (Kyoto, Japan). Fetal bovine serum (FBS), MagneGST Glutathione 32 3

Particles, Polyethylenimine (PEI)-Max, Nitrocellulose membrane, Chemiluminescence 34 36

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reagent, anti-FLAG-tag antibody, Block Ace, Can Get Signal Solutions, HiLyte Fluor 38

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555- and 647-conjugated secondary antibodies, Mounting medium, Anti-DDDDK-tag 40

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antibody agarose were obtained from Biosera (Nuaille, France), Promega (Madison, 42

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WI), Polysciences (Warrington, PA), Merck Millipore (Darmstadt, Germany), 43 4

Sigma-Aldrich (St. Louis, MO), Snow Brand Milk Products (Tokyo, Japan), TOYOBO 45 47

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(Osaka, Japan), AnaSpec (Fremont, CA), SeraCare Life Sciences (Milford, MA), and 49

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Medical and Biological Laboratories (Nagoya, Japan), respectively. All other chemicals 51

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and reagents were from common suppliers and were of the highest grade commercially 53

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available. 5

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Plasmids and protein expression 56 57 59

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Expression plasmid containing rat SNAP-25-B wild-type and C90A mutant 6 7

cDNA (SNAP-25-B/pcDNA3.2/nFLAG-DEST) were constructed as previously 8 9

described 19. 10

The human cplx-I entry vector (FLJ 92108AAAF) was kindly supplied

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by Dr. Naoki Goshima (National Institute of Advanced Industrial Science and 14

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Technology). Cplx-I was sub-cloned into pcDNA3.1/nV5-DEST or 16

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5’-FLAG-GST-tagged destination vectors (5FG-DEST) (kindly supplied by Dr. 18

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Goshima) by LR reaction. GST-tagged cplx-I was produced by a wheat germ cell-free 19 20

protein expression system (CellFree Science; Ehime, Japan), followed by purification 21 23

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using glutathione sepharose (GE Healthcare). 25

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Cell culture, transfection and 8-nitro-cGMP treatment 27

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SH-SY5Y cells were cultured in D-MEM supplemented with 10% FBS and 1% 29

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PS, and incubated in humidified atmosphere with 5% CO2 at 37℃. Cells were 30 31

transfected with plasmids using PEI-Max, incubated for 24 h, and then treated with 3

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8-nitro-cGMP for three hours 19. 34 36

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Western blot analysis 38

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Cells were lysed with phosphate buffered saline (PBS) containing 1% 40

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TritonX-100 and protease inhibitor cocktail. The cell lysates were subjected to 42

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SDS-PAGE, followed by western blotting. Immunoreactive bands were detected using 43 4

chemiluminescence reagent and luminescent image analyzer LAS-1000 (Fujifilm, 45 47

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Tokyo, Japan). 49

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Immunocytochemistry 51

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Immunocytochemistry was performed as described previously 12, 19. Briefly, 53

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after fixation, permeabilization, and blocking, the cells were incubated with primary 5

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antibodies overnight at 4℃, and then with fluorescent-conjugated secondary antibodies 56 57 59

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and Hoechst 33258. The images were captured and processed by using OLYMPUS 6 7

FV1200 IX83 microscope (OLUMPUS, Tokyo, Japan). 8 10

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LC-MS/MS analysis 12

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8-Nitro-cGMP in the SH-SY5Y cells were measured by LC-MS/MS using a 14

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stable-isotope dilution method as previously described13, 19. In brief, cells were lysed in 16

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methanol containing 1 µM 8- nitro -c[13C10]GMP and 5 mM NEM. After centrifugation, 18

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the supernatants were dried, resolved in 0.1% formic acid, and injected to high 19 20

performance liquid chromatography (HPLC) system (Waters, Milford, MA). Samples 21 23

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were separated by reverse-phase HPLC and analyzed with electrospray ionization triple 25

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quadrupole mass spectrometer (Xevo TQD; Waters,). The observed ion masses (parent 27

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→ daughter ions) were m/z 391→151 and m/z 401→156 for endogenous 29

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8-nitro-cGMP and spiked 8-nitro- c[13C10]GMP, respectively. 30 31

Pull-down assay 3

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Synaptosome was prepared as described previously19. This study was 34 36

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performed in accordance with the Guidelines for Animal Experimentation of Osaka 38

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Prefecture University. Synaptosome or SH-SY5Y cells transfected with FLAG-tagged 40

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SNAP-25 was treated with 8-nitro-cGMP for one or three hours, respectively. Purified 42

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GST-cplx bound to GSH-conjugated magnetic beads was incubated with the lysates for 43 4

three hours at room temperature. After washing, proteins bound to beads were eluted 45 47

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with SDS sample buffer and analyzed by western blotting. 49

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Co-immunoprecipitation (Co-IP) 51

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SH-SY5Y cells co-transfected with FLAG-tagged SNAP-25 and V5-tagged 53

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cplx were treated with 8-nitro-cGMP for three hours. Supernatants after centrifugation 5

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of cell lysates were incubated with anti-DDDDK-tag antibody-conjugated agarose for 56 57 59

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one hour at room temperature. After washing, proteins bound to beads were eluted with 6 7

SDS sample buffer. FLAG-tagged SNAP-25 and V5-tagged cplx were analyzed by 8 10

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western blotting. 12

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Blue-native (BN)-PAGE 14

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Native SNARE complex associated with cplx was analyzed by BN-PAGE as 16

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previously described29 with minor modifications. In brief, sample preparation was 18

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carried out using NativePAGETM Sample Prep Kit (Thermo) according to the 19 20

manufacture instructions. 8-Nitro-cGMP-treated SH-SY5Y cells-transfected with 21 2

V5-tagged cplx was lysed with lysis buffer containing NativePAGETM 1X Sample 25

24

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Buffer, 0.5% TritonX-100, 10% glycerol and protease inhibitors. Just after adding 27

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one-fourth volume of 0.5% CBB-G250 to samples, BN-PAGE was performed using 4% 29

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upper 4-16% gradient separation Bis-Tris polyacrylamide gel. After BN-PAGE, the 30 31

separation gels were soaked in transfer buffer containing 0.1 % SDS. followed by 32 3

transfer buffer containing 0.02% SDS for 10 min at room temperature, respectively. 34 36

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Proteins were transfer to PVDF membrane, CBB was removed by washing with 38

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methanol. V5-tagged cplx and SNAP-25 was detected by western blotting. 40

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Statistical Analysis. 42

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The data presented as the mean ± SE of individual experiments performed at least three 43 4

times. Statistical significance was determined by one-way analysis of variance 45 47

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(ANOVA), Tukey’s multiple comparison post-hoc test using the GraphPad Prism 49

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Software (GraphPad Software, La Jolla, CA). 50 51 52 53 54 5 56 57 59

58 14


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5

Author information: 7 9

8

Author Contributions 10 1

Y.K., K.K., T.A. and H.I. designed the research. Y.K., K.K. and A.K. performed 12 14

13

experiments, analysis and interpreted the data. H.I. conducted the experiments. Y.K. and 15 17

16

H.I. wrote the manuscript. 18 19 20 21 23

2

Funding Sources 24 26

25

This work was supported in part by a Grant-in-Aid for a Grant-in-Aid for Scientific 27 29

28

Research A (25253020 to T.A.), a Grant-in-Aid for Scientific Research B (16H04674 to 30 32

31

H.I.), a Grant-in-Aid for Challenging Exploratory Research (16K15208 to T.A. and 3 35

34

16K13089 to H.I.), and a Grant-in-Aid for Scientific Research on Innovative Areas 36 37

(Research in a Proposed Area) (26111008 to T.A. and 26111011 to H.I.) from the 38 39 40

Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. 41 42 43 4 46

45

Conflict of Interest 47 49

48

The authors declare no competing financial interest. 50 51 52 53 5

54

Acknowledgement 56 57 59

58 15



1 2 3 4 6

5

The authors thank Dr. Hidemitsu Nakajima (Osaka Prefecture University) for kindly 7 8

gifted us the cells and Dr. Naoki Goshima (National Institute of Advanced Industrial 9 10 1

Science and Technology) for kindly gifted us the plasmids. 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 59

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N., Itakura, M., Takahashi, M., Akaike, T., and Ihara, H. (2015) 8-Nitro-cGMP enhances SNARE complex formation through S-guanylation of Cys90 in SNAP25, ACS Chem. Neurosci. 6, 1715-1725. 20. Brunger, A. T. (2005) Structure and function of SNARE and SNARE-interacting proteins, Q. Rev. Biophys. 38, 1-47. 4

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Miyaoka, H., and Takahashi, M. (2015) Epileptogenesis and epileptic maturation in phosphorylation site-specific SNAP-25 mutant mice, Epilepsy Res. 115, 30-44. 23. Yamamori, S., Sugaya, D., Iida, Y., Kokubo, H., Itakura, M., Suzuki, E., Kataoka, M., Miyaoka, H., and Takahashi, M. (2014) Stress-induced phosphorylation of SNAP-25, Neurosci. Lett. 561, 182-187. 24. Mohrmann, R., Dhara, M., and Bruns, D. (2015) Complexins: small but capable, 56

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exhibit marked deficits in social behaviours but appear to be cognitively normal, Hum. Mol. Genet. 16, 2288-2305. 15

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27. Glynn, D., Drew, C. J., Reim, K., Brose, N., and Morton, A. J. (2005) Profound 16

ataxia in complexin I knockout mice masks a complex phenotype that includes exploratory and habituation deficits, Hum. Mol. Genet. 14, 2369-2385. 28. Wittig, I., Braun, H. P., and Schagger, H. (2006) Blue native PAGE, Nat. Protoc. 1, 418-428. 2

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29. Ramos-Miguel, A., Beasley, C. L., Dwork, A. J., Mann, J. J., Rosoklija, G., Barr, A. M., and Honer, W. G. (2015) Increased SNARE protein-protein interactions in orbitofrontal and anterior cingulate cortices in schizophrenia, Biol. Psychiatry 78, 27

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361-373. 30. Shata, A., Saisu, H., Odani, S., and Abe, T. (2007) Phosphorylated synaphin/complexin found in the brain exhibits enhanced SNARE complex binding, Biochem. Biophys. Res. Commun. 354, 808-813. 32

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FIGURE LEGENDS 6 7 9

8

Figure 1 Cell permeability of 8-nitro-cGMP in SH-SY5Y cells 10 12

1

SH-SY5Y cells were treated with 8-nitro-cGMP for three hours. (A) S-guanylated 14

13

proteins in SH-SY5Y cells were analyzed by western blotting using anti-S-guanylated 16

15

protein antibody. (B) 8-Nitro-cGMP and S-guanylated proteins in SH-SY5Y cells were 17 18

analyzed by immunohistochemical analysis. SH-SY5Y cells treated with 8-nitro-cGMP 19 21

20

were stained using anti-8-nitro-cGMP and anti-S-guanylated protein antibodies. DIC; 23

2

Differential Interference Contrast microscope. Scale bars; 100 µm. (C) 8-Nitro-cGMP 25

24

in SH-SY5Y cell was analyzed by LC-MS/MS analysis. 8-Nitro-cGMP in SH-SY5Y 27

26

cell was measured by LC-MS/MS using a stable-isotope dilution method. 29

28

Representative LC-MS/MS chromatograms were shown here. 30 31 32 3 35

34

Figure 2 Effects of 8-nitro-cGMP on interaction between SNARE complex and 37

36

externally added GST-cplx in synaptosomes and SH-SY5Y cells 38 40

39

Effects of 8-nitro-cGMP on interaction between SNARE complex and externally added 41 42

GST-cplx in synaptosomes and SH-SY5Y cells were analyzed by pull down assay. 43

(A)

45

4

Rat synaptosomes were treated with 8-nitro-cGMP (0, 10 or 100 µM). SNAP-25 47

46

pulled-down by GST-cplx was analyzed by western blotting using anti-FLAG-tag 49

48

antibody. (B) Graph shows bands intensity of pulled-down SNAP-25, normalized with 51

50

input data and presented as a percent of control (mean ± S.E.M.; n = 4). One-way 53

52

ANOVA with Tukey’s multiple comparison post-hoc test was used for statistical 5

54

analysis. *P < 0.05. (C) SH-SY5Y cells transfected with FLAG-tagged SNAP-25 56 57 59

58 20


Page 21 of 27


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4

(wild-type or C90A mutant) were treated with 8-nitro-cGMP (0, 10 or 100 µM). 6 7

FLAG-tagged SNAP-25 pulled-down by GST-cplx was analyzed by western blotting 8 10

9

using anti-FLAG-tag antibody. (D) Graph shows bands intensity of pulled-down 12

1

FLAG-tagged SNAP-25 normalized with input data and presented as a percent of 14

13

control (mean ± S.E.M.; n = 4). Student’s t-test and one-way ANOVA with Tukey’s 16

15

multiple comparison post-hoc test were used for statistical analysis. *P < 0.05, **P < 18

17

0.01 compared to the each control, ##P < 0.01 compared to the wild-type treated with 19 20

100 µM of 8-nitro-cGMP. 21 2 23 24 25 27

26

Figure 3 Effects of 8-nitro-cGMP on interaction between SNARE complex and 29

28

endogenously expressing V5-tagged cplx in SH-SY5Y cells 30 31 3

32

Effects of 8-nitro-cGMP on interaction between SNARE complex and endogenously 35

34

expressing V5-tagged cplx in SH-SY5Y cells were analyzed by co-IP assay. (A) 37

36

SH-SY5Y cells co-transfected with FLAG-tagged SNAP-25 (wild-type or C90A 39

38

mutant) and V5-tagged cplx were treated with 8-nitro-cGMP (0, 10 or 100 µM). 41

40

Immunoprecipitated FLAG-tagged SNAP-25 and co-immunoprecipitated V5-tagged 42 43

cplx were analyzed by western blotting using anti-FLAG-tag and anti-V5-tag antibodies, 4 46

45

respectively. (B) Graph shows bands intensity of co-immunoprecipitated V5-tagged 48

47

cplx normalized with immnoprecipitated FLAG-tagged SNAP-25 and presented as a 50

49

percent of control (mean ± S.E.M.; n = 6). Student’s t-test and Kruskal-Wallis test with 52

51

Dunn’s multiple comparison post-hoc test were used for statistical analysis. *P < 0.05 54

53

compared to the control, ##P < 0.01 compared to the wild-type treated with 100 µM of 5 56

8-nitro-cGMP. 57 59

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Figure 4 Effects of 8-nitro-cGMP on SNARE complex formation and association of 1

10

V5-tagged cplx in SH-SY5Y cells 12 14

13

Effects of 8-nitro-cGMP on SNARE complex formation and association of V5-tagged 16

15

cplx in SH-SY5Y cells were analyzed by BN-PAGE. 17

(A, C) SH-SY5Y cells

18

transfected with V5-tagged cplx were treated with 8-nitro-cGMP (0, 10 or 100 µM). 19 21

20

Proteins were separated by BN-PAGE followed by western blotting using anti-SNAP-25 23

2

(A) and anti-V5-tag antibodies (C). (B, D) Graph shows bands intensity of SNAP-25 at 25

24

high molecular weight (higher than 150 kDa, indicated by bracket (B)) and V5-tagged 27

26

cplx detected at approximately 1,000 kDa (indicated by arrow (D)). 29

28

Data were

presented as a percent of control (mean ± S.E.M.; n = 9). Kruskal-Wallis test with 31

30

Dunn’s multiple comparison post-hoc test were used for statistical analysis. *P < 0.05, 32 3 ***

34

P < 0.001 compared to the control.

35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 59

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Graphical Table of Contents 18

17 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60

ACS Paragon Plus Environment


Figure.1

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A 18

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B

8-Nitro-cGMP [µM] 20

19

kDa 21

250 23

150 24

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0

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DIC

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8-Nitro-cGMP [µM]

100 75 26

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50 29 30

37 31 32 34

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0

25 35

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20 37

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15 38 39 40

Actin 41 42 43 4 45 46 47 48

C 50

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Spiked 8-nitro-c[13C10]GMP 5

m/z 401 → 15 56

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2×104 counts

57 58 59

5×102 counts

0 60

8-Nitro-cGMP [µM] m/z 391 → 151

100

4.5

5×102 counts

5.0

5.5

6.0

Retention time [min]


S-guanylated

8-Nitro-cGMP

Page 25 of 27


Figure.2

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A 13

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B

14 15

8-Nitro-cGMP [µM] 16 17 18

0

10

100

Input 19 20 21 2

Bound 23

125 SNAP-25 in the GST-cplx1 pulled down [% of control]

1

10

24 25 26

100

*

75 50 25

27

0 28

8-Nitro-cGMP [µM] 29 30

C

8-Nitro-cGMP [µM]

41

40

39

38

37

Wild-type

36

35

34

3

0

10

Input Bound

42 43 4

8-Nitro-cGMP [µM]

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C90A

47

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Input Bound

10

100

D

0

100

100

SNAP-25 in the GST-cplx1 pulled down [% of control]

32

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0

125

*

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##

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**

0

8-Nitro-cGMP [mM]

54 5 56 57 58 59 60


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10 Wild-type

100

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Figure.3 19

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A 28

27 29

36

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3

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31

Wild-type

30

8-Nitro-cGMP [µM]

IP: FLAG-tag 0

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IB: FLAG-tag IB: V5-tag

37 38 39

IP: FLAG-tag 40

8-Nitro-cGMP [µM] 41

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4

C90A

43

42

IB: FLAG-tag IB: V5-tag

47

0

100

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B Immunoprecipitated V5-cplx/ SNAP-25-FLAG [% of control]

26

25

150

##

125 100

*

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8-Nitro-cGMP [mM]

48 49 50 51 52 53 54 5 56 57 58 59 60


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10 Wild-type

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A 8

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SNARE complex (% of control)

12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27

100 75 50 25

0 8-Nitro-cGMP [µM] 28 29 30 31

**

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32 3 34 36

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C 38

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D

39 40 41 42 43 4

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Cplx-V5 (% of control)

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0 8-Nitro-cGMP [µM]


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8-Nitro-cGMP Attenuates the Interaction between SNARE Complex

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